Assays, methods and means for modulating e2f activity

ABSTRACT

P/CAF interacts with and acetylates E2F, this acetylation affecting ability of E2F to stimulate transcription. Interaction between P/CAF and E2F and acetylation of E2F by P/CAF are modulated, affecting ability of E2F to stimulate transcription, induction of S-phase in cells, oncogenicity of cells, and induction of apoptosis in cells. Agents are obtained for treatment of disorders of cell growth.

This application is the U.S. national phase of international applicationPCT/GB99/01571 filed 17 May 1999, which designated the U.S.PCT/GB99/01571 claims priority to GB Application No. 9810562.0 filed 15May 1998.

The present invention relates to screening methods, peptides, mimetics,and methods of use based on the surprising discovery andcharacterisation of an interaction between known proteins, and thusnumerous cellular processes of interest in therapeutic contexts. Theproteins in question are P/CAF and E2F, it further being shown hereinthat P/CAF acetylates E2F at residues affecting ability of E2F tostimulate transcription. Prior to the making of the present invention itwas not known that acetylation of E2F affects its activity, inparticular its transcriptional activation, nor that P/CAF interacts withand acetylates E2F, thereby, affecting transcriptional activation byE2F.

The DNA in the nucleus is wrapped around a histone-core which is aprotein complex involving the four histones H4, H3, H2B and H2A. ThisDNA-histone structure (nucleosome) is not compatible with geneexpression. Re-organisation of the nucleosome is required fortranscription factors and RNA polymerase to have access to the DNA fortranscription.

Acetylation of histones at specific lysine residues has been correlatedwith an increase in gene expression. This modification is thought tore-model the nucleosomes and therefore increase the transcription of agiven template DNA. In the last two years enzymes have been identifiedwhich carry out this acetylation of histones. There are now fourfamilies of enzymes with such activity. These are GCN5 and P/CAF(Brownell, et al (1996), Cell, 84: 843-831 and Yang, et al (1996),Nature, 382: 319-324), CBP and p300 (Bannister and Kouzarides (1996),Nature, 384: 641-643 and Ogryzko, et al (1996), Cell, 87: 953-959), SRC1and ACTR (Chen, et al (1997), Cell, 90: 569-580 and Spencer, et al(1997), Nature, 389: 194-198) and TAF250 (Mizzen, et al (1996), Cell,87: 1261-1270). The precise in vivo targets of these enzymes are notknown. In vitro experiments suggest that recombinant enzymes may havespecificity for distinct lysines within the same histone. Precisely howacetylation of a particular histone increases transcription is notknown.

Recently, evidence has been provided that some proteins other thanhistones are acetylated. The p53 transcription factor and the basaltranscription factors TFIIE and TFIIF have been shown to be acetylated(Imhof, et al (1997), Current Biology, 7: 689-692). In the case of p53it has been shown that acetylation increases the DNA binding capacity ofthe protein.

The present invention is based on the surprising discovery that P/CAFinteracts with and acetylates E2F and that the acetylated residues areimportant for E2F function.

Experimental work on this is described below and leads to variousaspects of the present invention in which there is provided formodulation of interaction between P/CAF and E2F, particularlyacetylation of E2F by P/CAF.

Various aspects of the present invention provide for the use of P/CAFand E2F, with or without DNA, in screening methods and assays for agentswhich modulate interaction between P/CAF and E2F, particularlyacetylation of E2F by P/CAF, and agents which modulate the ability ofE2F to stimulate transcription and/or the function of E2F in inductionof S-phase, oncogenicity and/or induction of apoptosis.

Identification of key residues in E2F acetylated by P/CAF may also beused in the design of peptide and non-peptidyl agents which modulate,particularly inhibit, acetylation of E2F by P/CAF or other acetylaseenzyme, as discussed further below.

Methods of obtaining agents able to modulate interaction between P/CAFand E2F include methods wherein a suitable end-point is used to assessinteraction in the presence and absence of a test substance. Assaysystems may be used to determine P/CAF acetylase activity and/or P/CAFinteraction with E2F and/or acetylation of E2F by one or more otheracetylases. For acetylation assays, full-length E2F, truncated portionsof E2F, or portions of E2F fused to other proteins (eg. GST), or asuitable variant or derivative of any of these may be used. Peptideacetylation assays may be developed using peptides that correspond tothe acetylated regions of E2F. The acetylation of any of the above maybe assayed by any of a variety of procedures such as discussed below andmay be adapted to high throughput screening approaches. Generally ofmost interest is modulation of the acetylation of E2F by P/CAF or otheracetylase. Detailed disclosure in this respect is included below. It isworth noting, however,, that combinatorial library technology providesan efficient way of testing a potentially vast number of differentsubstances for ability to modulate an interaction with and/or activityof a polypeptide. Such libraries and their use are known in the art, forall manner of natural products, small molecules and peptides, amongothers. The use of peptide libraries may be preferred in certaincircumstances.

Given the results reported herein on which the present invention isbased, activators and inhibitors of P/CAF-associated acetylase activityor other acetylase able to acetylate E2F may be identified andappropriate agents may be obtained, designed and used for any of avariety of purposes. Modulation of E2F function may be used for thecontrol of cell proliferation. E2F1 is known to be oncogenic (Johnson,et al (1994), PNAS: 91, 12823-12827 and Singh, et al (1994), EMBO: Vol13, No 14, 3329-3338) and is able to induce S-phase when microinjectedinto cells (Johnson, et al, Nature: 365, 349-352). The ubiquitousexpression of E2F makes it a good candidate target for modulation ofgrowth control in a number of neoplasias, tumours, cancer, psoriasis,arteriosclerosis and other hyper-proliferative disorders. Overexpressionof E2F1 can cause cell death (apoptosis) (Shan and Lee, Molecular andCellular Biology: Vol 14, No 12, 8166-8173) and modulation of E2Ffunction in accordance with the present invention may be used to induceapoptosis.

Thus, various methods and uses of modulators, which inhibit orpotentiate interaction of P/CAF and E2F, and modulators that affectacetylation of E2F, are provided as further aspects of the presentinvention. The purpose of disruption, interference with or modulation ofinteraction between P/CAF and E2F, particularly the acetylation of E2Fby P/CAF may be to modulate any activity mediated by virtue of suchinteraction, as discussed above and further below.

Acetylation of E2F by one or more other acetylases may be modulated forthe same or similar purposes.

The full amino acid sequence of the P/CAF protein has been elucidatedand is set out in Yang, et al, Nature: 382, 319-324. Residues 352-658 ofP/CAF have been shown to include P/CAF histone acetyltransferaseactivity. Within this region is a stretch of about 100 residueshomologous to CBP (another histone acetyltransferase). Point mutationswithin P/CAF which eliminate the acetyltransferase activity aredisclosed in Martinez-Balbás, et al, EMBO: Vol 17, 101-108.

The E2F/DP family of transcription factors play a key role in regulatingthe mammalian cell cycle. They activate genes required for S-phase andin doing so can ultimately promote cell proliferation. Both E2F and DPfamily members have been shown to be oncogenic and E2F1 has beendemonstrated to be a potent inducer of S-phase (Lam, E. W-F. et al.Current Opinion in Cell Biology 1994, 6: 859-866).

The transcription activation capacity (and hence the oncogenicity) ofthe E2F/DP family is kept in check by the Retinoblastoma tumoursuppressor family of proteins (RB, p107, 0130). Members of this familybind to a transcriptional activation domain within the E2F protein. Bydoing so, the RB protein family members can silence the transcriptionalactivation capacity of the E2F/DP proteins and thus cause arrest in theG1-phase of the cell cycle. Release of RB from E2F/DP results in S-phaseinduction. This release is mediated by phosphorylation events (on RB andE2F) carried out by cyclin/CDK complexes towards the end of the G1 phase(Whyte, P. The retinoblastoma protein and its relatives. Seminars inCancer Biology 1995, 6: 83-90).

There are five identified members of the E2F family (E2F1-5) and threemembers of the DP family (DP1-3). All E2F members can form heterodimerswith all DP members. These heterodimers can bind and transactivate thepromoters of S-phase genes.

The E2F and DP proteins share a common class of DNA binding anddimerisation domain which allows them to form heterodimers and bind“E2F” binding sites co-operatively. Outside the DNA binding/dimerisationdomain the E2F family members have other sequences in common. They allhave a highly conserved “marked box”, whose function is unknown, and atranscriptional activation domain at the C-terminus which contains thebinding site for the RB family of proteins. DP family of proteins do notpossess any similarity to E2F proteins outside the DNA binding domain.However, they do contain highly conserved sequences which define thisfamily.

The activity of the various E2F/DP heterodimers comes from thetranscriptional activity of the E2F partner. No activation functionshave been attributed to DP proteins. The activation capacity of theE2F/DP complexes is negatively regulated by different members of the RBfamily: RB binds and represses E2F1-3 whereas p107 and p130 can bind andrepress E2F4 and E2F5. Brehm et al. (1998) Nature 391: 597-601 andMagnaghi-Jaulin et al. (1998) Nature 391: 601-605 show that theRetinoblastoma protein Rb recruits histone deacetylase to E2F,cooperating with the histone deacetylase to repress transcription fromE2F-regulated promoters.

An agent capable of modulating interaction between P/CAF and E2F may becapable of blocking interaction between P/CAF and one or more of thelysine residues in the various E2F's as follows, the alignment providingindication of significant homology between E2F1, E2F2 and E2F3:

Reference to acetylation of E2F generally applies herein unless contextrequires otherwise to any of the E2F's, but particularly E2F1, E2F2and/or E2F3, and most particularly E2F1.

In addition to interacting at the site of acetylation of E2F, P/CAF andE2F may interact at one or more other sites within either or bothproteins. Affecting interaction at such a site may have an effect onacetylation of E2F by P/CAF. Various fragments and derivatives of theproteins, particularly of E2F, may be used to analyse this, usingtechniques such as alanine scanning and deletion analysis. The presentinvention encompasses modulation of interaction between P/CAF and E2F atany site, preferably resulting in modulation of E2F acetylation.

Other agents according to the present invention useful in modulatingacetylation of E2F and therefore one or more of its functions modulatethe acetyltransferase activity of the acetylase. Such agents mayspecifically inhibit the ability of P/CAF to acetylate E2F. Assays andscreens for such agents are provided in accordance with the presentinvention, along with the agents themselves and their use in modulatingE2F acetylation and in modulating E2F function.

Agents useful in accordance with the present invention may be identifiedby screening techniques which involve determining whether an agent undertest inhibits or disrupts the interaction of P/CAF protein or a suitablefragment thereof (e.g. including amino acid residues of the acetylasedomain, or a smaller fragment of any of these regions) of P/CAF, withE2F or a fragment thereof, or a suitable analogue, fragment or variantthereof.

Suitable fragments of P/CAF or E2F include those which include residueswhich interact with the counterpart protein. Smaller fragments, andanalogues and variants of this fragment may similarly be employed, e.g.as identified using techniques such as deletion analysis or alaninescanning.

Thus, the present invention provides a peptide fragment of P/CAF whichis able to interact with E2F and/or inhibit interaction between P/CAFand E2F, particularly acetylation of E2F by P/CAF, and provides apeptide fragment of E2F which is able to interact with P/CAF and/orinhibit interaction between E2F and P/CAF, particularly acetylation ofE2F by P/CAF, such peptide fragments being obtainable by means ofdeletion analysis and/or alanine scanning of the relevant protein—makingan appropriate mutation in sequence, bringing together a mutatedfragment of one of the proteins with the other or a fragment thereof anddetermining interaction, preferably acetylation of E2F or fragmentthereof. In preferred embodiments, the peptide is short, as discussedbelow, and may be a minimal portion that is able to interact with therelevant counterpart protein and/or inhibit the relevant interaction.The invention further provides peptide fragments of E2F which are ableto inhibit acetylation of E2F at the relevant residues.

Other proteins may bind E2F at an acetylation site, and may bind or notdepending on whether the site is acetylated or not. A protein other thanP/CAF may bind at any one or more of the relevant lysines when they areacetylated, but not when not acetylated. A protein other than P/CAF maybind at any one or more of the relevant lysines when they are notacetylated, but not when acetylated. Such proteins may be identifiedusing standard methodology to identify interacting proteins. Forinstance, non-acetylated E2F fragments may be used in two-hybrid screensand chemically acetylated peptides may be screened against peptide andprotein libraries. The invention further extends to the use of E2F andpeptide fragments thereof including one or more of the relevant lysines,acetylated or not acetylated, for obtaining a peptide or protein (otherthan P/CAF) which binds at an acetylation site, particular a peptide orprotein which binds or not depending on whether the site is acetylatedor not. Further aspects of the invention provide assay methods for suchpeptides and proteins based on determining binding to E2F or a peptidefragment thereof, acetylated or not acetylated at one or more of therelevant lysines. The invention further extends to assays for substancesable to modulate interaction of such peptides or proteins with therelevant acetylation site, and to methods of modulating suchinteraction, also modulating agents.

Peptides in accordance with the present invention tend to be short, andmay be about 40 amino acids in length or less, preferably about 35 aminoacids in length or less, more preferably about 30 amino acids in length,or less, more preferably about 25 amino acids or less, more preferablyabout 20 amino acids or less, more preferably about 15 amino acids orless, more preferably about 10 amino acids or less, or 9, 8, 7, 6, 5 orless in length. Peptides according to the present invention may be about10-40 amino acids in length, about 5-10, about 10-15, about 10-20, about10-30, about 20-30, or about 30-40 amino acids in length. Peptides whichare E2F fragments may include one or more of the relevant lysineresidues noted above (e.g. for E2F1 Lys117, Lys120 and/or Lys125).

The present invention also encompasses peptides which are sequencevariants or derivatives of a wild type P/CAF or E2F sequence, but whichretain ability to interact with E2F or P/CAF (respectively, as the casemay be) and/or ability to modulate interaction between P/CAF and E2F,particularly acetylation of E2F by P/CAF, and/or ability to modulateacetylation of E2F by one or more other acetylases.

Instead of using a wild-type P/CAF or E2F fragment, a peptide orpolypeptide may include an amino acid sequence which differs by one ormore amino acid residues from the wild-type amino acid sequence, by oneor more of addition, insertion, deletion and substitution of one or moreamino acids. Thus, variants, derivatives, alleles, mutants andhomologues, e.g. from other organisms, are included.

Preferably, the amino acid sequence shares homology with a fragment ofthe relevant P/CAF or E2F fragment sequence shown preferably at leastabout 30%, or 40%, or 50%, or 60%, or 70%, or 75%, or 80%, or 85%, 90%or 95% homology. Thus, a peptide fragment of P/CAF or E2F may include 1,2, 3, 4, 5, greater than 5, or greater than 10 amino acid alterationssuch as substitutions with respect to the wild-type sequence.

A derivative of a peptide for which the specific sequence is disclosedherein may be in certain embodiments the same length or shorter than thespecific peptide. In other embodiments the peptide sequence or a variantthereof may be included in a larger peptide, as discussed above, whichmay or may not include an additional portion of P/CAF or E2F. 1, 2, 3, 4or 5 or more additional amino acids, adjacent to the relevant specificpeptide fragment in P/CAF or E2F, or heterologous thereto may beincluded at one end or both ends of the peptide.

As is well-understood, homology at the amino acid level is generally interms of amino acid similarity or identity. Similarity allows for“conservative variation”, i.e. substitution of one hydrophobic residuesuch as isoleucine, valine, leucine or methionine for another, or thesubstitution of one polar residue for another, such as arginine forlysine, glutamic for aspartic acid, or glutamine for asparagine.Similarity may be as defined and determined by the TBLASTN program, ofAltschul et al. (1990) J. Mol. Biol. 215: 403-10, which is in standarduse in the art, or more preferably using the algorithm GAP (GeneticsComputer Group, Madison, Wis.). GAP uses the Needleman and Wunschalgorithm to align two complete sequences that maximizes the number ofmatches and minimizes the number of gaps. Generally, the defaultparameters are used, with a gap creation penalty=12 and gap extensionpenalty=4. Use of either of the terms “homology” and “homologous” hereindoes not imply any necessary evolutionary relationship between comparedsequences, in keeping for example with standard use of terms such as“homologous recombination” which merely requires that two nucleotidesequences are sufficiently similar to recombine under the appropriateconditions. Homology may be over the full-length of the relevantpolypeptide or may more preferably be over a contiguous sequence ofabout 15, 20, 25, 30, 40, 50, 75, 100 or more amino acids, compared withthe relevant wild-type amino acid sequence.

At the nucleic acid level sequence identity may be assessed by means ofhybridization of molecules under stringent conditions. The presentinvention extends to nucleic acid that hybridizes with any one or moreof the specific sequences disclosed herein under stringent conditions.Suitable conditions include, e.g. for detection of sequences that areabout 80-90% identical, hybridization overnight at 42° C. in 0.25MNa₂HPO₄, pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 55°C. in 0.1×SSC, 0.1% SDS. For detection of sequences that are greaterthan about 90% identical, suitable conditions include hybridizationovernight at 65° C. in 0.25M Na₂HPO₄, pH 7.2, 6.5% SDS, 10% dextransulfate and a final wash at 60° C. in 0.1×SSC, 0.1% SDS.

As noted, variant peptide sequences and peptide and non-peptideanalogues and mimetics may be employed, as discussed further below.

Various aspects of the present invention provide a substance, which maybe a single molecule or a composition including two or more components,which includes a peptide fragment of P/CAF, particularly within theP/CAF acetylase domain, or E2F, particularly within the acetylatedregion of E2F, a peptide consisting essentially of such a sequence, apeptide including a variant, derivative or analogue sequence, or anon-peptide analogue or mimetic which has the ability to interact withP/CAF or E2F and/or modulate, disrupt or interfere with interactionbetween P/CAF and E2F.

Variants include peptides in which individual amino acids can besubstituted by other amino acids which are closely related as isunderstood in the art and indicated above.

Non-peptide mimetics of peptides are discussed further below.

As noted, a peptide according to the present invention and for use invarious aspects of the present invention may include or consistessentially of a fragment of P/CAF or E2F respectively. Where one ormore additional amino acids are included, such amino acids may be fromP/CAF or E2F or may be heterologous or foreign to P/CAF or E2F. Apeptide may also be included within a larger fusion protein,particularly where the peptide is fused to a non-P/CAF or non-E2F (i.e.heterologous or foreign) sequence, such as a polypeptide or proteindomain.

The invention also includes derivatives of the peptides, including thepeptide linked to a coupling partner, e.g. an effector molecule, alabel, a drug, a toxin and/or a carrier or transport molecule, and/or atargeting molecule such as an antibody or binding fragment thereof orother ligand. Techniques for coupling the peptides of the invention toboth peptidyl and non-peptidyl coupling partners are well known in theart. In one embodiment, the carrier molecule is a 16 aa peptide sequencederived from the homeodomain of Antennapedia (e.g. as sold under thename “Penetratin”), which can be coupled to a peptide via a terminal Cysresidue. The “Penetratin” molecule and its properties are described inWO 91/18981.

Peptides may be generated wholly or partly by chemical synthesis. Thecompounds of the present invention can be readily prepared according towell-established, standard liquid or, preferably, solid-phase peptidesynthesis methods, general descriptions of which are broadly available(see, for example, in J. M. Stewart and J. D. Young, Solid Phase PeptideSynthesis, 2nd edition, Pierce Chemical Company, Rockford, Ill. (1984),in M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis,Springer Verlag, New York (1984); and Applied Biosystems 430A UsersManual, ABI Inc., Foster City, Calif.), or they may be prepared insolution, by the liquid phase method or by any combination ofsolid-phase, liquid phase and solution chemistry, e.g. by firstcompleting the respective peptide portion an& then, if desired andappropriate, after removal of any protecting groups being present, byintroduction of the residue X by reaction of the respective carbonic orsulfonic acid or a reactive derivative thereof.

Another convenient way of producing a peptidyl molecule according to thepresent invention (peptide or polypeptide) is to express nucleic acidencoding it, by use of nucleic acid in an expression system.

Accordingly the present invention also provides in various aspectsnucleic acid encoding the polypeptides and peptides of the invention.

Generally, nucleic acid according to the present invention is providedas an isolate, in isolated and/or purified form, or free orsubstantially free of material with which it is naturally associated,such as free or substantially free of nucleic acid flanking the gene inthe human genome, except possibly one or more regulatory sequence(s) forexpression. Nucleic acid may be wholly or partially synthetic and mayinclude genomic DNA, cDNA or RNA. Where nucleic acid according to theinvention includes RNA, reference to the sequence shown should beconstrued as reference to the RNA equivalent, with U substituted for T.

Nucleic acid sequences encoding a polypeptide or peptide in accordancewith the present invention can be readily prepared by the skilled personusing the information and references contained herein and techniquesknown in the art (for example, see Sambrook, Fritsch and Maniatis,“Molecular Cloning, A Laboratory Manual, Cold Spring Harbor LaboratoryPress, 1989, and Ausubel et al, Short Protocols in Molecular Biology,John Wiley and Sons, 1992), given the nucleic acid sequence and clonesavailable. These techniques include (i) the use of the polymerase chainreaction (PCR) to amplify samples of such nucleic acid, e.g. fromgenomic sources, (ii) chemical synthesis, or (iii) preparing cDNAsequences. DNA encoding P/CAF or E2F fragments may be generated and usedin any suitable way known to those of skill in the art, including bytaking encoding DNA, identifying suitable restriction enzyme recognitionsites either side of the portion to be expressed, and cutting out saidportion from the DNA. The portion may then be operably linked to asuitable promoter in a standard commercially available expressionsystem. Another recombinant approach is to amplify the relevant portionof the DNA with suitable PCR primers. Modifications to the P/CAF or E2Fsequences can be made, e.g. using site directed mutagenesis, to lead tothe expression of modified P/CAF or E2F peptide or to take account ofcodon preference in the host cells used to express the nucleic acid.

In order to obtain expression of the nucleic acid sequences, thesequences can be incorporated in a vector having one or more controlsequences operably linked to the nucleic acid to control its expression.The vectors may include other sequences such as promoters or enhancersto drive the expression of the inserted nucleic acid, nucleic acidsequences so that the polypeptide or peptide is produced as a fusionand/or nucleic acid encoding secretion signals so that the polypeptideproduced in the host cell is secreted from the cell. Polypeptide canthen be obtained by transforming the vectors into host cells in whichthe vector is functional, culturing the host cells so that thepolypeptide is produced and recovering the polypeptide from the hostcells or the surrounding medium. Prokaryotic and eukaryotic cells areused for this purpose in the art, including strains of E. coli, yeast,and eukaryotic cells such as COS or CHO cells.

Thus, the present invention also encompasses a method of making apolypeptide or peptide (as disclosed), the method including expressionfrom nucleic acid encoding the polypeptide or peptide (generally nucleicacid according to the invention). This may conveniently be achieved bygrowing a host cell in culture, containing such a vector, underappropriate conditions which cause or allow expression of thepolypeptide. Polypeptides and peptides may also be expressed in in vitrosystems, such as reticulocyte lysate. Systems for cloning and expressionof a polypeptide in a variety of different host cells are well known.Suitable host cells include bacteria, eukaryotic cells such as mammalianand yeast, and baculovirus systems. Mammalian cell lines available inthe art for expression of a heterologous polypeptide include Chinesehamster ovary cells, HeLa cells, baby hamster kidney cells, COS cellsand many others. A common, preferred bacterial host is E. coli.

Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorfragments, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate. Vectors may be plasmids, viral e.g.‘phage, or phagemid, as appropriate. For further details see, forexample, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrooket al., 1989, Cold Spring Harbor Laboratory Press. Many known techniquesand protocols for manipulation of nucleic acid, for example inpreparation of nucleic acid constructs, mutagenesis, sequencing,introduction of DNA into cells and gene expression, and analysis ofproteins, are described in detail in Current Protocols in MolecularBiology, Ausubel et al. eds., John Wiley & Sons, 1992.

Thus, a further aspect of the present invention provides a host cellcontaining heterologous nucleic acid as disclosed herein.

The nucleic acid of the invention may be integrated into the genome(e.g. chromosome) of the host cell. Integration may be promoted byinclusion of sequences which promote recombination with the genome, inaccordance with standard techniques. The nucleic acid may be on anextra-chromosomal vector within the cell, or otherwise identifiablyheterologous or foreign to the cell.

A still further aspect provides a method which includes introducing thenucleic acid into a host cell. The introduction, which may (particularlyfor in vitro introduction) be generally referred to without limitationas “transformation”, may employ any available technique. For eukaryoticcells, suitable techniques may include calcium phosphate transfection,DEAE-Dextran, electroporation, liposome-mediated transfection andtransduction using retrovirus or other virus, e.g. vaccinia or, forinsect cells, baculovirus. For bacterial cells, suitable techniques mayinclude calcium chloride transformation, electroporation andtransfection using bacteriophage. As an alternative, direct injection ofthe nucleic acid could be employed.

Marker genes such as antibiotic resistance or sensitivity genes may beused in identifying clones containing nucleic acid of interest, as iswell known in the art.

The introduction may be followed by causing or allowing expression fromthe nucleic acid, e.g. by culturing host cells (which may include cellsactually transformed although more likely the cells will be descendantsof the transformed cells) under conditions for expression of the gene,so that the encoded polypeptide (or peptide) is produced. If thepolypeptide is expressed coupled to an appropriate signal leader peptideit may be secreted from the cell into the culture medium. Followingproduction by expression, a polypeptide or peptide may be isolatedand/or purified from the host cell and/or culture medium, as the casemay be, and subsequently used as desired, e.g. in the formulation of acomposition which may include one or more additional components, such asa pharmaceutical composition which includes one or more pharmaceuticallyacceptable excipients, vehicles or carriers (e.g. see below)

Introduction of nucleic acid encoding a peptidyl molecule according tothe present invention may take place in vivo by way of gene therapy, todisrupt or interfere with interaction between P/CAF and E2F or otherwiseaffect E2F acetylation.

Thus, a host cell containing nucleic acid according to the presentinvention, e.g. as a result of introduction of the nucleic acid into thecell or into an ancestor of the cell and/or genetic alteration of thesequence endogenous to the cell or ancestor (which introduction oralteration may take place in vivo or ex vivo), may be comprised (e.g. inthe soma) within an organism which is an animal, particularly a mammal,which may be human or non-human, such as rabbit, guinea pig, rat, mouseor other rodent, cat, dog, pig, sheep, goat, cattle or horse, or whichis a bird, such as a chicken. Genetically modified or transgenic animalsor birds comprising such a cell are also provided as further aspects ofthe present invention.

This may have a therapeutic aim. (Gene therapy is discussed below.)Also, the presence of a mutant, allele, derivative or variant sequencewithin cells of an organism, particularly when in place of a homologousendogenous sequence, may allow the organism to be used as a model intesting and/or studying substances which modulate activity of theencoded polypeptide in vitro or are otherwise indicated to be oftherapeutic potential. Knock-out mice, for instance, may be used to testfor radiosensitivity. Conveniently, however, at least preliminary assaysfor such substances may be carried out in vitro, that is within hostcells or in cell-free systems.

Where an effect of a test compound is established on cells in vitro,those cells or cells of the same or similar type may be grafted into anappropriate host animal for in vivo testing.

For instance, E2F function or activity may be measured in an animalsystem such as a tumour model, e.g. involving a xenograft, relying onactive E2F.

Suitable screening methods are conventional in the art. They includetechniques such as radioimmunosassay, scintillation proximetry assay andELISA methods. Suitably either the P/CAF protein or fragment or E2F orfragment, or an analogue, derivative, variant or functional mimeticthereof, is immobilised whereupon the other is applied in the presenceof the agents under test. In a scintillation proximetry assay abiotinylated protein fragment may be bound to streptavidin coatedscintillant—impregnated beads (produced by Amersham). Binding ofradiolabelled peptide is then measured by determination of radioactivityinduced scintillation as the radioactive peptide binds to theimmobilized fragment. Agents which intercept this are thus inhibitors ofthe interaction. Further ways and means of screening for agents whichmodulate interaction between P/CAF and E2F are discussed below.

In one general aspect, the present invention provides an assay methodfor an agent with ability to modulate, e.g. disrupt or interfere withinteraction between P/CAF and E2F, the method including:

(a) bringing into contact a first substance including a peptide fragmentof P/CAF or a derivative, variant or analogue thereof as disclosed, asecond substance including the relevant fragment of E2F or a variant,derivative or analogue thereof, and a test compound under conditions inwhich, in the absence of the test compound being an inhibitor, the firstand second substances interact; and

(b) determining interaction between the first and second substances.

A test compound which disrupts, reduces, interferes with or wholly orpartially abolishes interaction between said substances (e.g. includinga P/CAF fragment and including a E2F fragment), and which may modulateP/CAF and/or E2F activity, may thus be identified.

Agents which increase or potentiate interaction between the twosubstances may be identified using conditions which, in the absence of apositively-testing agent, prevent the substances interacting. As noted,such agents may be used to potentiate E2F function, for instance ininducing apoptosis.

Another general aspect of the present invention provides an assay methodfor a substance able to interact with the relevant region of P/CAF orE2F as the case may be, the method including:

(a) bringing into contact a substance which includes a peptide fragmentof P/CAF which interacts with E2F, or which includes a peptide fragmentof E2F which interacts with P/CAF, or a variant, derivative or analogueof such peptide fragment, as disclosed, and a test compound; and

(b) determining interaction between said substance and the testcompound.

A test compound found to interact with the relevant portion of P/CAF maybe tested for ability to modulate, e.g. disrupt or interfere with, P/CAFinteraction with E2F and/or ability to affect E2F and/or P/CAF activityor other activity mediated by P/CAF or E2F as discussed already above.

Similarly, a test compound found to interact with the relevant portionof E2F may be tested for ability to modulate, e.g. disrupt or interferewith, E2F interaction with P/CAF and/or ability to affect P/CAF and/orE2F activity or other activity mediated by E2F or P/CAF as discussedelsewhere herein.

Another general aspect of the present invention provides an assay methodfor a substance able to affect E2F activity, the method including:

(a) bringing into contact E2F and a test compound; and

(b) determining E2F activity (e.g. ability to activate transcriptionfrom an appropriate promoter, ability to induce S-phase, or ability toinduce apoptosis).

E2F activity may be determined in the presence and absence of P/CAF toallow for an effect of a test compound on activity to be attributed toan effect on interaction between E2F and P/CAF, preferably acetylationof E2F by P/CAF (discussed further below).

Assays for E2F transcriptional activation are standard in the art. Seee.g. Van Der Eb and Graham, Methods Enzymol: 65, 826-839.

The precise format of an assay of the invention may be varied by thoseof skill in the art using routine skill and knowledge. For example,interaction between substances may be studied in vitro by labelling onewith a detectable label and bringing it into contact with the otherwhich has been immobilised on a solid support. Suitable detectablelabels, especially for peptidyl substances include ³⁵S-methionine whichmay be incorporated into recombinantly produced peptides andpolypeptides. Recombinantly produced peptides and polypeptides may alsobe expressed as a fusion protein containing an epitope which can belabelled with an antibody.

The protein which is immobilized on a solid support may be immobilizedusing an antibody against that protein bound to a solid support or viaother technologies which are known per se. A preferred in vitrointeraction may utilise a fusion protein includingglutathione-S-transferase (GST). This may be immobilized on glutathioneagarose beads. In an in vitro assay format of the type described above atest compound can be assayed by determining its ability to diminish theamount of labelled peptide or polypeptide which binds to the immobilizedGST-fusion polypeptide. This may be determined by fractionating theglutathione-agarose beads by SDS-polyacrylamide gel electrophoresis.Alternatively, the beads may be rinsed to remove unbound protein and theamount of protein which has bound can be determined by counting theamount of label present in, for example, a suitable scintillationcounter.

An assay according to the present invention may also take the form of anin vivo assay. The in vivo assay may be performed in a cell line such asa yeast strain or mammalian cell line in which the relevant polypeptidesor peptides are expressed from one or more vectors introduced into thecell.

The ability of a test compound to modulate interaction between P/CAF andE2F may be determined using a so-called two-hybrid assay.

For example, a polypeptide or peptide containing a fragment of P/CAF orE2F as the case may be, or a peptidyl analogue or variant thereof asdisclosed, may be fused to a DNA binding domain such as that of theyeast transcription factor GAL 4. (A particularly preferred fragment ofP/CAF may include or be the acetylase domain or a fragment of theacetylase domain.) The GAL 4 transcription factor includes twofunctional domains. These domains are the DNA binding domain (GAL4DBD)and the GAL4 transcriptional activation domain (GAL4TAD). By fusing onepolypeptide or peptide to one of those domains and another polypeptideor peptide to the respective counterpart, a functional GAL 4transcription factor is restored only when two polypeptides or peptidesof interest interact. Thus, interaction of the polypeptides or peptidesmay be measured by the use of a reporter gene probably linked to a GAL 4DNA binding site which is capable of activating transcription of saidreporter gene. This assay format is described by Fields and Song, 1989,Nature 340; 245-246. This type of assay format can be used in bothmammalian cells and in yeast. Other combinations of DNA binding domainand transcriptional activation domain are available in the art and maybe preferred, such as the LexA DNA binding domain and the VP60transcriptional activation domain.

When looking for peptides or other substances which interfere withinteraction between a P/CAF polypeptide or peptide and E2F polypeptideor peptide, the P/CAF or E2F polypeptide or peptide may be employed as afusion with (e.g.) the LexA DNA binding domain, and the counterpart E2For P/CAF polypeptide or peptide as a fusion with (e.g.) VP60, andinvolves a third expression cassette, which may be on a separateexpression vector, from which a peptide or a library of peptides ofdiverse and/or random sequence may be expressed. A reduction in reportergene expression (e.g. in the case of β-galactosidase a weakening of theblue colour) results from the presence of a peptide which disrupts theP/CAF/E2F interaction, which interaction is required for transcriptionalactivation of the β-galactosidase gene. Where a test substance is notpeptidyl and may not be expressed from encoding nucleic acid within asaid third expression cassette, a similar system may be employed withthe test substance supplied exogenously.

When performing a two hybrid assay to look for substances whichinterfere with the interaction between two polypeptides or peptides itmay be preferred to use mammalian cells instead of yeast cells. The sameprinciples apply and appropriate methods are well known to those skilledin the art.

In preferred assays according to the present invention, the end-point ofthe assay, that is to say that which is determined in order to assessthe effect of the test agent on the interaction of interest, isacetylation of E2F or a fragment, variant or derivative thereof.

Thus, a further aspect of the present invention provides an assay methodincluding:

(a) bringing into contact a substance which includes at least a fragmentof P/CAF which acetylates E2F, a substance which includes at least afragment of E2F including a site acetylated by P/CAF, and a testcompound; and

(b) determining acetylation at said site.

Of course, any suitable variant or derivative of P/CAF and/or E2F may beemployed in such an assay and any suitable fragments of E2F may beemployed including any of the sites of acetylation, such as includingone or more of the relevant lysines as discussed above (e.g. for E2F1lys117, lys120 and/or lys125).

Another aspect of the present invention provides an assay method for asubstance able to affect E2F acetylation, the method including:

(a) treating acetylated E2F with a test compound; and

(b) determining acetylation of the E2F.

A still further aspect of the present invention provides an assay methodfor a substance able to affect E2F acetylation, the method including:

(a) treating with a test compound E2F which is not acetylated at one ormore of the relevant positions noted above; and

(b) determining acetylation of the E2F.

As noted, E2F may be acetylated at one or more residues, particularlyone or more of the lysine residues which correspond to Lys117, Lys120and/or Lys125 in E2F1.

Acetylation may be determined for example by immobilising E2F or afragment, variant or derivative thereof, e.g. on a bead or plate, anddetecting acetylation using an antibody or other binding molecule whichbinds the relevant site of acetylation with a different affinity whenthe site is acetylated from when the site is not acetylated. Suchantibodies may be obtained by means of any standard technique asdiscussed elsewhere herein, e.g. using a acetylated peptide (such as afragment of E2F). Binding of a binding molecule which discriminatesbetween the acetylated and non-acetylated form of E2F or relevantfragment, variant or derivative thereof may be assessed using anytechnique available to those skilled in the art, which may involvedetermination of the presence of a suitable label.

Acetylation may also be assayed in solution, e.g. as described inBannister and Kouzarides (1996), Nature, 384: 641-643. Briefly, proteinsubstrate (˜1 μg) and ˜0.1 pmol of acetyltransferase are mixed to give afinal volume of 30 μl in buffer IPH (50 mM Tris.HCl pH8.0, 150 mM NaCl,5 mM EDTA, 0.5% [v/v] NP-40, 0.1 mM PMSF). Reactions are initiated bythe addition of [14-C]-acetyl coA (1.85 kBq: 1.85 GBq/mmol; Amersham)and incubated at 30° C. for 10-45 min. The reaction products are thenresolved by SDS-PAGE and viewed following fluorography of the gel.Alternatively, following SDS-PAGE, the resolved proteins can be Westernblotted to a nitrocellulose membrane, which is then dried and exposed tofilm.

A further option is an in-gel activity assay, such as described byBrownell and Allis (1995), Proc. Natl. Acad. Sci., 92: 6364-6368 orMizzen, et al (1996), Cell, 87: 1261-1270. Samples may be crude cellularextracts, partially purified fractions, highly purified cellularproteins or bacterially produced and purified recombinant proteins.Before loading onto the activity gel the sample is made to 1×SDS-PAGloading buffer and boiled for 2 minutes. The gel is a standard LaemmliSDS-PAG except that purified protein substrate is added to the resolvinggel to a final concentration of 1 mg/ml. Polymerisation of the gel isinitiated using standard techniques, at which point the proteinsubstrate becomes immobilized within the gel matrix. After adding thestacking gel, the samples are loaded and the gel run as a standardSDS-PAG. After the gel has run it is soaked, with gentle agitation, in100 ml of wash buffer (50 mM Tris.HCl pH8.0, 0.1% β-mercaptoethanol)containing 20% (v/v) isopropanol for 20 minutes at room temperature.This washing step is repeated twice. Proteins in the gel are thendenatured by washing in 100 ml of wash buffer containing 8M urea for 20minutes at room temperature. This denaturing step is repeated twice. Thegel is then soaked without agitation in 100 ml of wash buffer containing0.04% tween-31 40 for 20 minutes at 4° C. This step is then repeated butfor a duration of 12 hours, after which the gel is washed twice for aperiod of 20 minutes each time. After the final soak the gel/buffer isallowed to slowly come to room temperature. The gel is washed in washbuffer containing 10% (v/v) glycerol for 20 minutes at room temperature.The gel is then placed in a heat sealable bag and 3 ml of the samebuffer containing 10 μCi of [3H]-acetylCoA is added. The contents arethoroughly mixed, air bubbles removed and the bag sealed. The reactionis then performed by immersing the bag in a 30° C. water-bath for atleast 30 minutes. Following the acetylation step, the gel is recoveredand washed extensively in several 100 ml changes of gel destain solution(10% [v/v] methanol, 10% [v/v] acetic acid). This washing stage isperformed at room temperature with agitation and should include anovernight wash.

An agent able to inhibit acetylation of E2F by P/CAF or other acetylasemay include or other substance able to affect the catalytic propertiesof the enzymatically active site of the acetylase. An inhibitor ofacetylation may interact with P/CAF or other acetylase within theacetylase domain. Residues within this domain are involved withinteraction with E2F and catalysis of the acetylation. Residues outsideof the domain may also be involved in interacting with E2F and agentswhich interfere with such interaction may affect the acetylation asdiscussed elsewhere herein.

Nucleic acid constructs in which a site recognised by E2F and at which,on binding when acetylated, E2F stimulates transcription from anoperably-linked promoter, may be used to assess the effect a testsubstance has on E2F function, by determination of promoter activity.E2F binding sites for different members of the family have beenestablished by PCR selection assays (Tao, et al, Molecular and CurrentBiology: Vol 17, No 12, 6994-7007. An example of a suitable promoter isthat of the human cyclin E gene, a relevant biological target of E2F1.There is evidence that activation of this promoter by E2F1 is requiredby for cell cycle progression (Ohtani, et al, PNAS: 92, 12146-12150 andBotz, et al, Molecular and Cellular Biology: Vol 16, No 7, 3401-3409).(Agents in accordance with the present invention may be found whichaffect cyclin E transcription and cell cycle progression, and may beused to affect these.)

Thus a further assay method according to the present invention involves

(a) providing E2F or a fragment, variant or analogue thereof able toactivate transcription from a promoter including a E2F binding site, atest compound, and a reporter construct including a promoter whichincludes a E2F binding site and which is operably linked to a reportersequence for transcription thereof, under conditions wherein, in theabsence of the test compound being an inhibitor of E2F acetylation, thereporter sequence is transcribed;

(b) determining promoter activity.

P/CAF and/or other acetylase able to acetylate E2F may be included inthe assay medium.

A further assay method of the present invention includes

(a) providing E2F or a fragment thereof which is not acetylated at oneor more of the relevant positions noted above, a test compound, and areporter construct including a promoter which includes a E2F bindingsite and which is operably linked to a reporter sequence fortranscription thereof, under conditions wherein if the test compoundpromotes acetylation of E2F the reporter sequence is transcribed;

(b) determining promoter activity.

P/CAF and/or other acetylase able to acetylate E2F may be included inthe assay medium.

A further assay method according to the present invention includes

(a) providing E2F or a fragment, variant or analogue thereof able tointeract with P/CAF and able to activate transcription from a promoterincluding a E2F binding site, P/CAF or a fragment, variant or analoguethereof able to interact with E2F, a test compound, and a reporterconstruct including a promoter which includes a E2F binding site andwhich is operably linked to a reporter sequence for transcriptionthereof, under conditions wherein, in the absence of the test compoundbeing an inhibitor of interaction between P/CAF and E2F, the reportersequence is transcribed;

(b) determining promoter activity.

The test compound may promote interaction between P/CAF and E2F andenhance transcription of the reporter sequence.

The interaction between P/CAF and E2F may include acetylation of E2F.

“Promoter activity” is used to refer to ability to initiatetranscription. The level of promoter activity is quantifiable forinstance by assessment of the amount of mRNA produced by transcriptionfrom the promoter or by assessment of the amount of protein productproduced by translation of mRNA produced by transcription from thepromoter. The amount of a specific MRNA present in an expression systemmay be determined for example using specific oligonucleotides which areable to hybridise with the mRNA and which are labelled or may be used ina specific amplification reaction such as the polymerase chain reaction.Use of a reporter gene facilitates determination of promoter activity byreference to protein production.

In such a construct, the promoter is operably linked to a gene, e.g. acoding sequence. Generally, the gene may be transcribed into mRNA whichmay be translated into a peptide or polypeptide product which may bedetected and preferably quantitated following expression. A gene whoseencoded product may be assayed following expression is termed a“reporter gene”, i.e. a gene which “reports” on promoter activity.

The reporter gene preferably encodes an enzyme which catalyses areaction which produces a detectable signal, preferably a visuallydetectable signal, such as a coloured product. Many examples are known,including β-galactosidase and luciferase. β-galactosidase activity maybe assayed by production of blue colour on substrate, the assay being byeye or by use of a spectrophotometer to measure absorbance.Fluorescence, for example that produced as a result of luciferaseactivity, may be quantitated using a spectrophotometer. Radioactiveassays may be used, for instance using chloramphenicolacetyltransferase, which may also be used in non-radioactive assays. Thepresence and/or amount of gene product resulting from expression fromthe reporter gene may be determined using a molecule able to bind theproduct, such as an antibody or fragment thereof. The binding moleculemay be labelled directly or indirectly using any standard technique.

Those skilled in the art are well aware of a multitude of possiblereporter genes and assay techniques which may be used to determine geneactivity. Any suitable reporter/assay may be used and it should beappreciated that no particular choice is essential to or a limitation ofthe present invention.

Thus, nucleic acid constructs comprising a promoter and a reporter genemay be employed in screening for a substance able to modulate thetranscriptional activator activity of E2F on the promoter. Fortherapeutic purposes, e.g. for treatment of cancer, a substance able toinhibit expression of the promoter, i.e. antagonise the stimulatorfunction of E2F, may be sought. A method of screening for ability of asubstance to modulate activity of E2F may comprise contacting anexpression system, such as a host cell, containing a nucleic acidconstruct as discussed with a test or candidate substance anddetermining expression of the reporter gene. The level of expression inthe presence of the test substance may be compared with the level ofexpression in the absence of the test substance. A difference inexpression in the presence of the test substance may indicate ability ofthe substance to modulate E2F function.

A promoter construct may be introduced into a cell line using anytechnique previously described to produce a stable cell line containingthe reporter construct integrated into the genome. The cells may begrown and incubated with test compounds for varying times. The cells maybe grown in 96 well plates to facilitate the analysis of large numbersof compounds. The cells may then be washed and the reporter geneexpression analysed. For some reporters, such as luciferase the cellswill be lysed then analysed.

Preliminary assays in vitro may be followed by, or run in parallel with,in vivo assays.

Of course, the person skilled in the art will design any appropriatecontrol experiments with which to compare results obtained in testassays.

Performance of an assay method according to the present invention may befollowed by isolation and/or manufacture and/or use of a compound,substance or molecule which tests positive for ability to modulateinteraction between P/CAF and E2F and/or modulate P/CAF or E2F activityor a mediated activity. Following identification of a suitable agent itmay be investigated further. Furthermore, it may be manufactured and/orused in preparation, i.e. manufacture or formulation, of a compositionsuch as a medicament, pharmaceutical composition or drug. These may beadministered to individuals.

The amount of test substance or compound which may be added to an assayof the invention will normally be determined by trial and errordepending upon the type of compound used. Typically, from about 0.001 nMto 1 mM or more concentrations of putative inhibitor compound may beused, for example from 0.01 nM to 100 μM, e.g. 0.1 to 50 μM, such asabout 10 μM. Greater concentrations may be used when a peptide is thetest substance. Even a molecule which has a weak effect may be a usefullead compound for further investigation and development.

Compounds which may be used may be natural or synthetic chemicalcompounds used in drug screening programmes. Extracts of plants whichcontain several characterised or uncharacterised components may also beused.

Antibodies directed to the site of interaction in either protein form afurther class of putative inhibitor compounds.

Candidate inhibitor antibodies may be characterised and their bindingregions determined to provide single chain antibodies and fragmentsthereof which are responsible for disrupting the interaction. The term“antibody molecule” may generally be used to cover antibody fragments,derivatives, functional equivalents and homologues of antibodies,including synthetic molecules and molecules whose shape mimicks that ofan antibody enabling it to bind an antigen or epitope.

Example antibody fragments, capable of binding an antigen or otherbinding partner are the Fab fragment consisting of the VL, VH, Cl andCH1 domains; the Fd fragment consisting of the VH and CH1 domains; theFv fragment consisting of the VL and VH domains of a single arm of anantibody; the dAb fragment which consists of a VH domain; isolated CDRregions and F(ab′)2 fragments, a bivalent fragment including two Fabfragments linked by a disulphide bridge at the hinge region. Singlechain Fv fragments are also included.

As noted above, antibody molecules may be used for determining whetheror not a peptide or polypeptide (e.g. E2F or fragment thereof) isacetylated, provided the relevant antibody molecule is able todiscriminate between acetylated and non-acetylated forms of the peptide.

The reactivities of antibodies on a sample may be determined by anyappropriate means. Tagging with individual reporter molecules is onepossibility. The reporter molecules may directly or indirectly generatedetectable, and preferably measurable, signals. The linkage of reportermolecules may be directly or indirectly, covalently, e.g. via a peptidebond or non-covalently. Linkage via a peptide bond may be as a result ofrecombinant expression of a gene fusion encoding antibody and reportermolecule. The mode of determining binding is not a feature of thepresent invention and those skilled in the art are able to choose asuitable mode according to their preference and general knowledge.

Antibodies may also be used in purifying and/or isolating a polypeptideor peptide according to the present invention, for instance followingproduction of the polypeptide or peptide by expression from encodingnucleic acid therefor. Antibodies may be useful in a therapeutic context(which may include prophylaxis) to disrupt P/CAF and E2F interactionwith a view to inhibiting their activity. Antibodies can for instance bemicro-injected into cells, e.g. at a tumour site, subject to radio-and/or chemo-therapy (as discussed already above). Antibodies may beemployed in accordance with the present invention for other therapeuticand non-therapeutic purposes which are discussed elsewhere herein.

Other candidate inhibitor compounds may be based on modelling the3-dimensional structure of a polypeptide or peptide fragment and usingrational drug design to provide potential inhibitor compounds withparticular molecular shape, size and charge characteristics.

A compound found to have the ability to affect P/CAF and/or E2F activityhas therapeutic and other potential in a number of contexts, asdiscussed. For therapeutic treatment such a compound may be used incombination with any other active substance, e.g. for anti-tumourtherapy another anti-tumour compound or therapy, such as radiotherapy orchemotherapy. In such a case, the assay of the invention, when conductedin vivo, need not measure the degree of modulation of interactionbetween E2F and P/CAF (or appropriate fragment, variant or derivativethereof) or of modulation of E2F acetylation or activity caused by thecompound being tested. Instead the effect on transition of cells intoS-phase, oncogenesis in tissue culture and/or induction of cell death byapoptosis may be determined. It may be that such a modified assay is runin parallel with or subsequent to the main assay of the invention inorder to confirm that any such effect is as a result of the inhibitionof interaction between P/CAF and E2F caused by said inhibitor compoundand not merely a general toxic effect.

Thus, an agent identified using one or more primary screens (e.g. in acell-free system) as having ability to interact with P/CAF and/or E2Fand/or modulate activity of P/CAF and/or E2F may be assessed furtherusing one or more secondary screens. A secondary screen may involvetesting for a biological function of E2F as noted above (e.g. inductionof S-phase or apoptosis).

As noted, the agent may be peptidyl, e.g. a peptide which includes asequence as recited above, or may be a functional analogue of such apeptide.

As used herein, the expression “functional analogue” relates to peptidevariants or organic compounds having the same functional activity as thepeptide in question, which may interfere with the interaction betweenP/CAF and E2F. Examples of such analogues include chemical compoundswhich are modelled to resemble the three dimensional structure of theP/CAF or E2F domain in the contact area, and in particular thearrangement of the key amino acid residues as they appear in P/CAF orE2F.

In a further aspect, the present invention provides the use of the abovesubstances in methods of designing or screening for mimetics of thesubstances.

Accordingly, the present invention provides a method of designingmimetics of P/CAF or E2F having the biological activity of E2F or P/CAFbinding or inhibition, the activity of allosteric inhibition of E2F orP/CAF and/or the activity of modulating, e.g. inhibiting, P/CAF/E2Finteraction, said method comprising:

(i) analysing a substance having the biological activity to determinethe amino acid residues essential and important for the activity todefine a pharmacophore; and,

(ii) modelling the pharmacophore to design and/or screen candidatemimetics having the biological activity.

Suitable modelling techniques are known in the art. This includes thedesign of so-called “mimetics” which involves the study of thefunctional interactions fluorogenic oligonucleotide the molecules andthe design of compounds which contain functional groups arranged in sucha manner that they could reproduced those interactions.

The designing of mimetics to a known pharmaceutically active compound isa known approach to the development of pharmaceuticals based on a “lead”compound. This might be desirable where the active compound is difficultor expensive to synthesise or where it is unsuitable for a particularmethod of administration, e.g. some peptides may not be well suited asactive agents for oral compositions as they tend to be quickly degradedby proteases in the alimentary canal. Mimetic design, synthesis andtesting may be used to avoid randomly screening large number ofmolecules for a target property.

There are several steps commonly taken in the design of a mimetic from acompound having a given target property. Firstly, the particular partsof the compound that are critical and/or important in determining thetarget property are determined. In the case of a peptide, this can bedone by systematically varying the amino acid residues in the peptide,e.g. by substituting each residue in turn. These parts or residuesconstituting the active region of the compound are known as its“pharmacophore”.

Once the pharmacophore has been found, its structure is modelled toaccording its physical properties, e.g. stereochemistry, bonding, sizeand/or charge, using data from a range of sources, e.g. spectroscopictechniques, X-ray diffraction data and NMR. Computational analysis,similarity mapping (which models the charge and/or volume of apharmacophore, rather than the bonding between atoms) and othertechniques can be used in this modelling process.

In a variant of this approach, the three-dimensional structure of theligand and its binding partner are modelled. This can be especiallyuseful where the ligand and/or binding partner change conformation onbinding, allowing the model to take account of this the design of themimetic.

A template molecule is then selected onto which chemical groups whichmimic the pharmacophore can be grafted. The template molecule and thechemical groups grafted on to it can conveniently be selected so thatthe mimetic is easy to synthesise, is likely to be pharmacologicallyacceptable, and does not degrade in vivo, while retaining the biologicalactivity of the lead compound. The mimetic or mimetics found by thisapproach can then be screened to see whether they have the targetproperty, or to what extent they exhibit it. Further optimisation ormodification can then be carried out to arrive at one or more finalmimetics for in vivo or clinical testing.

The mimetic or mimetics found by this approach can then be screened tosee whether they have the target property, or to what extent theyexhibit it. Further optimisation or modification can then be carried outto arrive at one or more final mimetics for in vivo or clinical testing.

Mimetics of this type together with their use in therapy form a furtheraspect of the invention.

The present invention further provides the use of a peptide whichincludes a sequence as disclosed, or a derivative, active portion,analogue, variant or mimetic, thereof able to interact with P/CAF or E2Fand/or modulate, inhibit or potentiate, interaction between P/CAF andE2F and/or modulate, inhibit or potentiate, P/CAF and/or E2F activity,in screening for a substance able to interact with E2F and/or P/CAF,and/or modulate, inhibit or potentiate, interaction between P/CAF andE2F, and/or modulate P/CAF and/or E2F activity.

Generally, such a substance according to the present invention isprovided in an isolated and/or purified form, i.e. substantially pure.This may include being in a composition where it represents at leastabout 90% active ingredient, more preferably at least about 95%, morepreferably at least about 98%. Such a composition may, however, includeinert carrier materials or other pharmaceutically and physiologicalyacceptable excipients. As noted below, a composition according to thepresent invention may include in addition to an modulator compound asdisclosed, one or more other molecules of therapeutic use, such as ananti-tumour agent.

The present invention extends in various aspects not only to a substanceidentified as a modulator of P/CAF and E2F interaction and/or P/CAF orE2F-mediated activity, property or pathway, in accordance with what isdisclosed herein, but also a pharmaceutical composition, medicament,drug or other composition comprising such a substance, a methodcomprising administration of such a composition to a patient, e.g. for apurpose discussed elsewhere herein, which may include preventativetreatment, use of such a substance in manufacture of a composition foradministration, e.g. for a purpose discussed elsewhere herein, and amethod of making a pharmaceutical composition comprising admixing such asubstance with a pharmaceutically acceptable excipient, vehicle orcarrier, and optionally other ingredients.

A substance according to the present invention such as an inhibitor ofP/CAF and E2F interaction may be provided for use in a method oftreatment of the human or animal body by therapy which affects an P/CAFor E2F-mediated activity in cells, e.g. tumour cells. Other purposes ofa method of treatment employing a substance in accordance with thepresent invention are discussed elsewhere herein.

Thus the invention further provides a method of modulating an P/CAFand/or E2F-mediated activity, e.g. for a purpose—discussed elsewhereherein, which includes administering an agent which modulates, inhibitsor blocks the interaction of P/CAF with E2F protein, such a method beinguseful in treatment where such modulation, inhibition or blocking isdesirable, or an agent which increase, potentiates or strengthensinteraction of P/CAF with E2F, useful in treatment where this isdesirable.

The invention further provides a method of treatment which includesadministering to a patient an agent which interferes with theinteraction of P/CAF with E2F. Exemplary purposes of such treatment arediscussed elsewhere herein.

Whether it is a polypeptide, antibody, peptide, nucleic acid molecule,small molecule, mimetic or other pharmaceutically useful compoundaccording to the present invention that is to be given to an individual,administration is preferably in a “prophylactically effective amount” ora “therapeutically effective amount” (as the case may be, althoughprophylaxis may be considered therapy), this being sufficient to showbenefit to the individual. The actual amount administered, and rate andtime-course of administration, will depend on the nature and severity ofwhat is being treated. Prescription of treatment, e.g. decisions ondosage etc, is within the responsibility of general practitioners andother medical doctors.

A composition may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated.

Pharmaceutical compositions according to the present invention, and foruse in accordance with the present invention, may include, in additionto active ingredient, a pharmaceutically acceptable excipient, carrier,buffer, stabiliser or other materials well known to those skilled in theart. Such materials should be non-toxic and should not interfere withthe efficacy of the active ingredient. The precise nature of the carrieror other material will depend on the route of administration, which maybe oral, or by injection, e.g. cutaneous, subcutaneous or intravenous.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may include a solid carriersuch as gelatin or an adjuvant. Liquid pharmaceutical compositionsgenerally include a liquid carrier such as water, petroleum, animal orvegetable oils, mineral oil or synthetic oil. Physiological salinesolution, dextrose or other saccharide solution or glycols such asethylene glycol, propylene glycol or polyethylene glycol may beincluded.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,Lactated Ringer's Injection. Preservatives, stabilisers, buffers,antioxidants and/or other additives may be included, as required.

Liposomes, particularly cationic liposomes, may be used in carrierformulations.

Examples of techniques and protocols mentioned above can be found inRemington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

The agent may be administered in a localised manner to a tumour site orother desired site or may be delivered in a manner in which it targetstumour or other cells.

Targeting therapies may be used to deliver the active agent morespecifically to certain types of cell, by the use of targeting systemssuch as antibody or cell specific ligands. Targeting may be desirablefor a variety of reasons, for example if the agent is unacceptablytoxic, or if it would otherwise require too high a dosage, or if itwould not otherwise be able to enter the target cells.

Instead of administering these agents directly, they may be produced inthe target cells by expression from an encoding gene introduced into thecells, eg in a viral vector (a variant of the VDEPT technique—seebelow). The vector may targeted to the specific cells to be treated, orit may contain regulatory elements which are switched on more or lessselectively by the target cells.

The agent (e.g. small molecule, mimetic) may be administered in aprecursor form, for conversion to the active form by an activating agentproduced in, or targeted to, the cells to be treated. This type ofapproach is sometimes known as ADEPT or VDEPT, the former involvingtargeting the activator to the cells by conjugation to a cell-specificantibody, while the latter involves producing the activator, e.g. anenzyme, in a vector by expression from encoding DNA in a viral vector(see for example, EP-A-415731 and WO 90/07936).

An agent may be administered in a form which is inactive but which isconverted to an active form in the body. For instance, the agent may bephosphorylated (e.g. to improve solubility) with the phosphate beingcleaved to provide an active form of the agent in the body.

A composition may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated, such as cancer, virus infection or any othercondition in which a P/CAF or E2F-mediated effect is desirable.

Nucleic acid according to the present invention, encoding a polypeptideor peptide able to modulate, e.g. interfere with, P/CAF and E2Finteraction and/or induce or modulate activity or other P/CAF orE2F-mediated cellular pathway or function, may be used in methods ofgene therapy, for instance in treatment of individuals, e.g. with theaim of preventing or curing (wholly or partially) a disorder or foranother purpose as discussed elsewhere herein.

Vectors such as viral vectors have been used in the prior art tointroduce nucleic acid into a wide variety of different target cells.Typically the vectors are exposed to the target cells so thattransfection can take place in a sufficient proportion of the cells toprovide a useful therapeutic or prophylactic effect from the expressionof the desired polypeptide. The transfected nucleic acid may bepermanently incorporated into the genome of each of the targeted cells,providing long lasting effect, or alternatively the treatment may haveto be repeated periodically.

A variety of vectors, both viral vectors and plasmid vectors, are knownin the art, see U.S. Pat. No. 5,252,479 and WO 93/07282. In particular,a number of viruses have been used as gene transfer vectors, includingpapovaviruses, such as SV40, vaccinia virus, herpesviruses, includingHSV and EBV, and retroviruses. Many gene therapy protocols in the priorart have used disabled murine retroviruses.

As an alternative to the use of viral vectors other known methods ofintroducing nucleic acid into cells includes electroporation, calciumphosphate co-precipitation, mechanical techniques such asmicroinjection, transfer mediated by liposomes and direct DNA uptake andreceptor-mediated DNA transfer.

Receptor-mediated gene transfer, in which the nucleic acid is linked toa protein ligand via polylysine, with the ligand being specific for areceptor present on the surface of the target cells, is an example of atechnique for specifically targeting nucleic acid to particular cells.

A polypeptide, peptide or other substance able to modulate or interferewith the interaction of the relevant polypeptide, peptide or othersubstance as disclosed herein, or a nucleic acid molecule encoding apeptidyl such molecule, may be provided in a kit, e.g. sealed in asuitable container which protects its contents from the externalenvironment. Such a kit may include instructions for use.

Various further aspects and embodiments of the present invention will beapparent to those skilled in the art in view of the present disclosure.Certain aspects and embodiments of the invention will now be illustratedby way of example.

All documents mentioned anywhere herein are incorporated by reference.

EXPERIMENTAL

Experiment 1

An important regulator of the cell cycle, the E2F1 transcription factor,was found to be acetylated in vitro by the P/CAF acetyltransferase.

Recombinant GST-E2F-1 or BSA or GST proteins were incubated with³HAcetylCoA in the presence or absence (−) of P/CAF and CBP, during 30min. at 30° C. After that the reaction products were loaded in a 10% PAAgel, this was fluorographed, dried and exposed over night. In the lanecorresponding to GST-E2F-1 incubation with P/CAF a band corresponding toacetylated GST-E2F-1 protein was seen, but not in any other of thelanes.

Experiment 2

A series of deletions of human E2F1 were used to show acetylation of theprotein in the region between amino acids 89 and 287.

Different GST-E2F-1 deletion proteins (the portions of E2F correspondingto amino acids 380-432, 358-432, 287-432 and 89-432), as well as GST-EMAand GST-DP1 proteins were incubated with ³HAcetylCoA in the presence ofP/CAF for 30 min. at 30° C. The reaction products were loaded in a 105PAA gel, and this was fluorographed, dried and exposed over night. Aband corresponding to acetylated GST-E2F-1 protein was seen in only onelane, corresponding to incubation of the portion of E2F-1 of amino acids89-432, indication acetylation taking place between amino acids 89 and287 of E2F-1.

Experiment 3

A series of point mutations of human E2F1 were used to show theacetylation of three closely spaced lysines at position 117, 120 and 125has important biological significance. Mutation of all three lysines toarginine resulted in an E2F1 protein which can no longer be acetylatedby P/CAF.

Point mutations were made where Lys (161, 164), (182, 183, 185) and(117, 120, 125) on GST-E2F-1 were changed to alanines, using a sitedirected mutagenesis Kit from Stratagene. The mutated proteins wereexpressed in E. Coli and subjected to acetylation by incubating themwith P/CAF in the presence of ³HAcetylCoA for 30 min. at 30° C. Afterthat they were separated in a 10% PAA gel, this was fluorographed, driedand exposed over night at −70° C. A band corresponding to acetylatedGST-E2F-1 protein was seen in all lanes except for that corresponding toincubation of E2F-1 K (117, 120, 125).

Experiment 4

Acetylation of E2F1 by P/CAF was shown to increase the ability of E2F1to activate transcription.

A reporter plasmid containing E2F binding sites upstream from a CATreporter was introduced into U205 cells along with wild-type E2F ormutant E2F in which lysines 117, 120 and 125 were changed to arginine.Each transfection also contained DP1 (which co-operates with E2F in DNAbinding).

When P/CAF was included in the transfection, the wild-type E2F wasstimulated in activity, but the mutant was not.

If a mutant P/CAF was included, mutated in the HAT domain to abolishacetyltransferase activity, the stimulation of wild-type E2F was notseen.

These results show that P/CAF stimulates the activation capacity of E2F,and that this is by virtue of the ability of P/CAF to acetylate E2F atlysine residues 117, 120 and 125.

Thus, preventing acetylation of E2F may be used to affect its biologicalfunction. Since E2F1, E2F2 and E2F3 are able to induce S-phase and celltransformation via the activation potential, disruption of E2Facetylation (in particular of E2F1, E2F2 or E2F3) may be used to inhibitcell proliferation. Also, given the role of E2F in inducing apoptosis,modulating E2F acetylation may be used to potentiate cell killing.

Experiment 5

By means of Western blotting using an antibody which recognisesaceylated lysine residues, E2F was shown to be acetylated in U205 cells.E2F was precipitated with an E2F-1 specific antibody and then Westernblotted with an anti-acetylated lysine antibody.

Experiment 6

Co-immunoprecipitation was used to show that P/CAF binds E2F1 in vivo.

P/CAF tagged with a Flag was transfected into cells. E2F-1 was thenimmunoprecipitated and the Flag-tagged P/CAF was detected by a Westernblot. No band was detected in the absence of P/CAF or when truncatedP/CAF (residues 352-832) was employed.

Immunoprecipitation Methodology

Cells were harvested by trypsinization, washed twice with ice-cold PBSand resuspended in 500 μl RIPA buffer (150 mM NaCl, 10.0 % NP-40, 0.5%DOC, 0.1% SDS, 50 mM Tris-HCl pH 8.0) supplemented with a cocktail ofprotease inhibitors (Complete, Boehringer Mannheim). After 30 minincubation on ice the whole cell extracts were clarified bycentrifugation at 12,000 g for 10 min, and the supernatant preclearedwith 30 μl protein A/G sepharose at 4° C. for 30 min. 2 μg of polyclonalanti-E2F1 antibody C20 (Santa Cruz) were added to the extracts androcked at 4° C. overnight. Then 30 μl protein A/G sepharose were addedfor another 2 hr at 4° C. After 4 extensive washes in RIPA buffer, thebeads were boiled in SDS containing sample buffer. Proteins wereseparated on a 12.5% SDS-PAGE and transferred to nitrocellulose membraneby standard procedures. For the detection of acetylated E2F1 in vivo,Western blot analysis was performed with an polyclonal anti-AcLysantibody. For pulse chase analysis, the blots were exposed to X-ray(Biomax MS, Kodak) and the intensity of [³⁵S] labeled E2F protein wasmeasured with the NIH Image 1.61 program and calculated in comparison tothe protein present at time zero.

Experiment 7

Acetylation of E2F by P/CAF was shown to lead to an increase in abilityof E2F to bind DNA, as determined by means of a DNA mobility-shift assayperformed using recombinant, bacterially expressed E2F and a DNAoligonucleotide with an E2F1 binding site.

For electrophoretic mobility shift assay (EMSA) His-tagged E2F1,GST-tagged E2F1 arginine mutant and GST-tagged DP1 were synthesized inE. coli, purified on Ni NTA-agarose or glutathione sepharose and elutedfrom the beads. E2F1 and E2F1 mutant were acetylated in IPH buffer inthe presence of 2 mM acetyl coA and GST-PCAF(352-658) protein and thenused for EMSA. 25 μl DNA binding reaction mixture contained bindingbuffer (50 mM KCl, 10 mM MgCl₂, 0.5 mM DTT, 20 mM HEPES pH 7.5), 100 ngpoly(dA)-poly(dT), 25 μg bovine serum albumine, 0.5 μg sonicated salmonesperm DNA, approximately 50-200 ng fusion proteins and 50 fmol³²P-labeled oligonucleotide (modified E2F1 binding site of DHFR genepromoter). Binding reactions were performed at room temperature for 10min. The reaction products were separated in a 4% polyacrylamid gel runin 0.25×TBE (22.5 mM Tris-borate, 0.5 mM EDTA). Subsequently the gel wasdried and exposed to X-ray.

Experiment 8

Experiment 7 was repeated except recombinant E2F was used in whichresidues 117, 120 and 125 had been changed to arginine.

Unlike the wild-type acetylated E2F1 (see Experiment 7), themutant—unacetylated—E2F1 was not stimulated by P/CAF in DNA binding.

Experiment 9

P/CAF was found to stabilised E2F protein. Introduction of P/CAF intocells was found to lead to the presence of elevated levels of E2F1, asseen by pulse chase labelling of E2F and Western blot. The pulse chaseshows that in the presence of P/CAF the E2F protein has a longerhalf-life.

293T cells were transfected overnight using the calcium phosphatemethod. 24 h after removal of the transfection precipitate, the cellswere washed twice with PBS and starved for 1 hr in methionine/cysteinefree medium. Subsequently, 0.2 mCi of [³⁵S]methionine/cystein (NEN) wasadded to each flask, and the cells were incubated for 2 hr. After thelabeling, the cells were harvested for time zero or incubated withnormal medium supplemented with 10 fold excess of nonradioactivemethionine and cysteine for 1, 3 and 5 hr and harvested thereafter.Equal amounts of radio active lysate were used for immunoprecipitationas described above.

The stabilisation was shown to be mediated by the acetyltransferaseactivity of P/CAF: a P/CAF mutant lacking active HAT domain (ΔHAT) didnot stabilise E2F. The stabilisation was also seen on introduction of RB(retinoblastoma protein), and effect which has been reported before andserves as a positive control.

1. An assay method for an agent which affects E2F acetylation, themethod including: (a) treating an acetylated E2F polypeptide or anacetylated E2F peptide with a test compound, or (b) treating with a testcompound an E2F polypeptide or an acetylated E2F peptide which comprisesone or more lysine residues corresponding to those found at positions117, 120 and 125 in wild-type E2F1, in which polypeptide or peptide oneor more of said lysines is not acetylated, or (c) bringing into contacta substance which includes a P/CAF polypeptide which acetylates E2F, asubstance which includes an E2F polypeptide or an E2F peptide includinga site acetylated by P/CAF, and a test compound; and, following step a,b or c, (d) determining acetylation of the E2F polypeptide or E2Fpeptide, wherein said E2F polypeptide has a sequence selected from thegroup consisting of the human E2F1, E2F2, E2F3, E2F4 and E2F5 sequences,said E2F peptide is a peptide fragment of a sequence selected from thegroup consisting of the human E2F1, E2F2, E2F3, E2F4 and E2F5 sequences,and; said P/CAF polypeptide has the sequence of human P/CAF.
 2. An assaymethod for an agent which affects E2F activity, the method including:(a) bringing into contact E2F and a test compound; and (b) determiningE2F activity in the presence and absence of a P/CAF polypeptide whichacetylates E2F, wherein E2F has a sequence selected from the groupconsisting of the human E2F1, E2F2, E2F3, E2F4 and E2F5 sequences andsaid P/CAF polypeptide has the sequence of human P/CAF.
 3. An assaymethod for an agent which affects E2F activity, the method comprising:(a) providing an E2F polypeptide which activates transcription from apromoter including an E2F binding site, a test compound, and a reporterconstruct including a promoter which includes an E2F binding site andwhich is operably linked to a reporter sequence for transcriptionthereof, under conditions wherein, in the absence of the test compoundbeing an inhibitor of E2F acetylation, the reporter sequence istranscribed, or (b) providing an E2F polypeptide which activatestranscription from a promoter including an E2F binding site, whichpolypeptide comprises one or more lysine residues corresponding to thosefound at positions 117, 120 and 125 in wild-type E2F1, and in whichpolypeptide or peptide one or more of said lysines is not acetylated, atest compound, and a reporter construct including a promoter whichincludes an E2F binding site and which is operably linked to a reportersequence for transcription thereof, under conditions wherein if the testcompound promotes acetylation of E2F the reporter sequence istranscribed, or (c) providing an E2F polypeptide which interacts withP/CAF and activates transcription from a promoter including an E2Fbinding site, a P/CAF polypeptide which interacts with E2F, a testcompound, and a reporter construct including a promoter which includesan E2F binding site and which is operably linked to a reporter sequencefor transcription thereof, under conditions wherein, in the absence ofthe test compound being an inhibitor of interaction between P/CAF andE2F, the reporter sequence is transcribed; and, following step a, b or c(d) determining promoter activity, wherein said E2F polypeptide has asequence selected from the group consisting of human E2F1, E2F2, E2F3,E2F4 and E2F5 sequence; and said P/CAF polypeptide has the sequence ofhuman P/CAF.
 4. An assay method for an agent which modulates interactionbetween P/CAF and E2F, the method including: (a) bringing into contact afirst substance including a P/CAF polypeptide or a P/CAF peptide, asecond substance including an E2F polypeptide or an E2F peptide, and atest compound under conditions in which, if of the test compound doesnot disrupt the interaction between P/CAF and E2F, the first and secondsubstances interact; and (b) determining interaction between the firstand second substances, wherein said E2F polypeptide has sequenceselected from the group consisting of the human E2F1, E2F2, E2F3, E2F4and E2F5 sequences; said E2F peptide is a peptide fragment of a sequenceselected from the group consisting of the human E2F1, E2F2, E2F3, E2F4and E2F5 sequences; and, said P/CAF polypeptide has the sequence ofhuman P/CAF.
 5. An assay method for an agent which affects one or moreof (i) ability of E2F to stimulate transcription, (ii) induction ofS-phase in cells, (iii) oncogenicity of cells, and/(iv) induction ofapoptosis in cells, the method comprising: (a) bringing into contact aP/CAF polypeptide and a test compound, and (b) determining P/CAFacetyltransferase activity; wherein a test compound which inhibits P/CAFacetyltransferase activity is identified as a candidate said agent,wherein E2F is selected from the group consisting of human E2F1, E2F2,E2F3, E2F4 and E2F5; and, said P/CAF polypeptide has the sequence ofhuman P/CAF.
 6. A method according to claim 5 comprising determiningacetylation of E2F by said P/CAF polypeptide.
 7. A method according toclaim 5 comprising determining E2F activity.
 8. A method according toclaim 5 wherein a test compound which inhibits P/CAF acetyltransferaseactivity is further tested for ability to affect one or more of (i)ability of E2F to stimulate transcription, (ii) induction of S-phase incells, (iii) oncogenicity of cells, and (iv) induction of apoptosis incells.
 9. An assay method for an agent which interacts with a region ofP/CAF or a region of E2F, which region of P/CAF interacts with E2F andwhich region of E2F interacts with P/CAF, a said agent which interactswith a said region being a candidate modulator of interaction betweenP/CAF and E2F, the method including: (a) bringing into contact asubstance which includes a P/CAF peptide which interacts with E2F, orwhich includes an E2F peptide which interacts with P/CAF, and a testcompound; and (b) determining interaction between said substance and thetest compound, wherein said E2F polypeptide has a sequence selected fromthe group consisting of the human E2F1, E2F2, E2F3, E2F4 and E2F5sequences; said E2F peptide is a peptide fragment of a sequence selectedfrom the group consisting of human E2F1, E2F2, E2F3, E2F4 and E2F5sequences; and, said P/CAF polypeptide has the sequence of human P/CAF.10. A method according to any one of claims 1, 2, 3, 4, 5 and 9 furthercomprising formulating a said agent into a composition comprising atleast one additional component.